Chimeric gene for the transformation of plants

ABSTRACT

1) Chimeric gone for conferring to plants an increased tolerance to a herbicide. 2) It comprises, in the direction of transcription, a promoter region, a transit peptide region, a sequence encoding glyphosate tolerance and a polyadenylation signal region, wherein the promoter region consists of at least one promoter of a plant histone gene enabling the expression of the herbicide tolerance protein in the regions of glyphosate accumulation. 3) Production of glyphosate-tolerant plants.

This application is a continuation of U.S. Ser. No. 08/475,189, filedJun. 7, 1995. Now U.S. Pat. No. 5,792,930 which is a continuation ofU.S. Ser. No. 08/239,947, filed on May 9, 1994, now U.S. Pat. No.5,491,288, which is a file wrapper continuation of U.S. Ser. No.07/847,597, filed May 5, 1992, now abandoned.

The present invention relates to novel promoters, to novel chimericgenes containing them and to their use in plants for conferring to theman increased tolerance to herbicides. It also relates to the plant cellstransformed by means of these genes and to the transformed plantsregenerated from these cells as well as to the plants derived fromcrossbreedings using these transformed plants.

Glyphosate, sulfosate or fosametine are broad- spectrum systemicherbicides of the phosphonomethyl-glycine family. They act essentiallyas competitive inhibitors of 5-(enolpyruvyl)shikimate-3-phosphatesynthase (EC 2.5.1.19) or EPSPS in relation to PEP(phosphoenolpyruvate). After their application to the plant, they aretranslocated inside the plant where they accumulate in the rapidlygrowing parts, in particular the caulinary and root apexes, causing thedeterioration and even the destruction of sensitive plants.

Plastidial EPSPS, the main target of these products, is an enzyme of thearomatic amino acid biosynthesis pathway which is encoded by one or morenuclear genes and synthesised in the form of a cytoplasmic precursor andthen imported into the plastids where it accumulates in its naturalform.

The tolerance of plants to glyphosate and to products of the family isobtained by the stable introduction inside their genome of an EPSPS geneof plant or bacterial origin mutant or nonmutant with respect to thecharacteristics of the inhibition of the product of this gene byglyphosate. Given the mode of action of glyphosate, it is useful to beable to express the product of translation of this gene so as to permitits substantial accumulation in plastids.

It is known, for example from American U.S. Pat. No. 4,535,060, toconfer to a plant a tolerance to a herbicide of the abovementioned type,in particular N-(phosphonomethyl)glycine or glyphosate, by introducinginto the plant genome a gene encoding an EPSPS carrying at least onemutation making this enzyme more resistant to its competitive inhibitor(glyphosate), after localisation of the enzyme in the plastidialcompartment. However, these techniques need to be improved in order toachieve greater reliability in the use of these plants under agronomicconditions.

In the present description, “plant” is understood as meaning anydifferentiated multicellular organism capable of photosynthesis and“plant cell” any cell derived from a plant and capable of formingundifferentiated tissues such as calluses or differentiated tissues suchas embryos or plant sections, plants or seeds.

The subject of the present invention is the production of transformedplants having an increased tolerance to herbicides in general andparticularly of the phosphonomethylglycine family by regenerating cellstransformed by means of novel chimeric genes comprising a gene fortolerance of these herbicides. The invention also relates to novelchimeric genes as well as to transformed plants which are more tolerantdue to better tolerance of the rapidly growing parts, as well as toplants derived from crossbreedings using these transformed plants. Thesubject of the invention is also novel promoters for constructing theabove chimeric genes and comprising a DNA sequence capable of serving aspromoter region in a chimeric gene which may be used for transformingplants, which comprises, in the direction of transcription, at least onepromoter or a fragment thereof of a plant histone gene enabling theexpression of the herbicide tolerance protein in the regions ofaccumulation of said herbicide.

More particularly, the subject of the invention is a chimeric gene forconferring to plants an increased tolerance to a herbicide whose targetis EPSPS, comprising, in the direction of transcription, a promoterregion, a transit peptide region, a sequence encoding a glyphosatetolerance enzyme and a polyadenylation signal region, wherein thepromoter region consists of at least one fragment of a plant histonegene promoter enabling the preferential expression of a herbicidetolerance protein in the regions of glyphosate accumulation.

The histone gene is derived from a monocotyledonous plant such as forexample wheat, maize or rice, or preferably from a dicotyledonous plantsuch as for example lucerne, sunflower, soya bean, colza or preferablyArabidopsis thaliana.CF Plant Mol. Biol Vol 8, 1987, p 179-191 Chabouteet al.

A histone gene of the H3 or preferably H4 type is preferably used, aloneor under a multiplicated especially duplicated, form.

The promoter region of the chimeric gene according to the invention mayin addition advantageously comprise at least one fragment of a promoterfrom a gene which is expressed naturally in plants, that is to say, forexample, a promoter of viral origin such as the 35S RNA promoter of thecauliflower mosaic virus (CaMv35S), or of plant origin such as the smallsubunit of the ribulose 1,5-diphosphate carboxylase oxygenase (RuBisCO)gene from a crop such as for example maize or sunflower.

The transit peptide region comprises, in the direction of transcription,at least one transit peptide of a plant gene encoding aplastid-localised enzyme, a partial sequence of the N-terminal maturepart of a plant gene encoding a plastid-localised enzyme and then asecond transit peptide of a plant gene encoding a plastid-localisedenzyme, preferably the RuBisCO gene.

The above transit peptides which can be used in the transit peptideregion may be known per se and may be of plant origin, for example,derived from maize, sunflower, peas, tobacco or the like. The first andthe second transit peptides may be identical, analogous or different.They may in addition each comprise one or more transit peptide units.

The partial sequence of the N-terminal mature part is derived from aplant gene encoding a plastid- localised enzyme, such as for example amaize, sunflower or pea gene or the like, it being possible for theoriginal plant species to be identical, analogous or different from thatfrom which the first and second transit peptides are derivedrespectively. Furthermore, the partial sequence of the mature part maycomprise a varying number of amino acids, generally from 15 to 40,preferably from 18 to 33.

Construction of the entire transit peptide region may be known per se,in particular by fusion or any other suitable means. The role of thischaracteristic region is to enable the release of a mature protein witha maximum efficiency, preferably in native form.

The coding sequence for herbicide tolerance which may be used in thechimeric gene according to the invention encodes a mutant EPSPS having adegree of glyphosate tolerance. This sequence, obtained in particular bymutation of the EPSPS gene, may be of bacterial origin, for examplederived from Salmonella typhymurium (and called in the text whichfollows “AroA gene”), or of plant origin, for example from petunia orfrom tomatoes. This sequence may comprise one or more mutations, forexample the Pro 101 to Ser mutation or alternatively the Gly 96 to Alamutations. The untranslated polyadenylation signal region in 3′ of thechimeric gene according to the invention may be of any origin, forexample bacterial, such as the nopaline synthase is gene, or of plantorigin, such as the small subunit of the maize or sunflower RuBisCO.

The chimeric gene according to the invention may comprise, in additionto the above essential parts, an untranslated intermediate region(linker) between the promoter region and the coding sequence which maybe of any origin, bacterial, viral or plant.

EXAMPLE 1

CONSTRUCTION OF A CHIMERIC GENE

The construction of a chimeric gene according to the invention iscarried out using the elements:

1) Promoter: the promoter is isolated from an H4 A777 histone clone anda H4 A748 histone clone respectively, from Arabidopsis thaliana,Strasbourg strain, which are described by Chaboute (thesis from theUniversity of Strasbourg, 1987) and PLANT MOL.BIOL. Vol 8, 1987 p179-191. These promoters, comprising about 1 kpb between the XhoI sitesused for its isolation, are used as they are or in duplicate form orfused with a single or double CaMV35S promoter, that is to say part ofwhich has been duplicated.

2) Transfer region: the two transit peptides as well as the matureprotein elements used are derived from the cloned cDNA of the smallsubunit of maize RuBisCO corresponding to the gene described by Lebrunet al. Nucl. Acids Res. 15:4360 (1987) and from the cloned cDNA of thesmall subunit of sunflower RuBisCO isolated by Waksman et al. Nucl.Acids Res. 15:1328 (1987). More specifically, the transfer regioncomprises, in the direction of translation:

a transit peptide of the small subunit of sunflower RuBisCO,

a sequence of 22 amino acids of the N-terminal mature part of the smallsubunit of maize RuBisCO,

a transit peptide of the small subunit of maize RuBisCO.

Other similar genes may contain sequences of 10 to 40 and preferably 18and 33 amino acids respectively.

In order to provide a comparative element, another construction wascarried out containing, in the direction of transcription, “doubleCaMV35S promoter/ transit peptide of the SSU of the sunflowerRuBisCO/N-terminal sequence of 22 amino acids of the SSU of maizeRuBisCO/transit peptide of the SSU of maize RuBisCO/AroA gene/nos”(pRPA-BL 410).

3) Structural gene: it is derived from the mutant (Pro 101 to Ser) EPSPSgene of Salmonella typhymurium isolated by Stalker et al. J. Biol. Chem.260:4724 (1985). The pMG34-2 clone (provided by Calgene) was linearizedwith XbaI and then treated with Vigna radiata nuclease. After recuttingwith SmaI, the two blunt ends were ligated. The clone obtained possessesan NcoI site in the initiator ATG as well as a 17-bp SalI sitedownstream of the stop codon. This clone was called pRPA-BL 104.

4) Polyadenylation signal region: the fragment is derived from thenopaline synthase gene of pTi37 (Bevan et al. Nature 304:187 , 1983).This site is contained in a 260-bp MboI fragment (Fraley et al., 1983;Patent Application PCT 84/02913) which was treated with Klenowpolymerase and cloned in the SmaI site of M13 mp 18 in order tointroduce the BamHI and EcoRI sites at the 5′ and 3′ ends respectively.

After cutting with BamHI and treating with Vigna radiata nucleasefollowed by cutting with EcoRI and treating with Klenow polymerase, theresulting fragment was introduced in the vector p-BL 20 (cf. FrenchPatent Application 88/04130), cut by XbaI and BamHI and treated withKlenow polymerase. After recutting with SalI and SstI, a fragment ofabout 0.4 kbp containing the 3′ nos sequence on the side of the SalIsite and the right end on the T-DNA side of the SstI site is obtained.

The assembly of all these elements was carried out in the followingmanner:

“Transit peptide of the SSU of the maize RuBisCO/AroA gene” fusion:

The transit peptide of the SSU of the maize RuBisCO gene is derived froma 192-bp EcoRI-SphI fragment obtained from the cDNA corresponding to theSSU gene of the maize RuBisCO gene, described by Lebrun et al. (1987),possessing an NcoI site spanning the initiation codon for translationand an SphI site corresponding to the cleavage site of the transitpeptide.

Translational fusion is obtained between the maize transit peptide andthe bacterial EPSPS gene by treating the SphI end with bacteriophage T4polymerase and by ligating it with the Klenow polymerase-treated NcoIend of the AroA gene from pRPA-BL 104, recut with EcoRI.

Transit peptide of the SSU of maize RuBisCO/sequence of 22 amino acidsof the mature part of the maize gene/AroA gene fusion:

Similarly, a 228-bp EcoRI-HindII fragment of the cDNA of the SSU of themaize RuBisCO gene is ligated with the Klenow polymerase-treated NcoIend of the AroA gene from pRPA-BL 104 and recut with EcoRI. Atranslational fusion is obtained between the transit peptide of the SSUof maize RuBisCO, the 22 amino acids of the mature part of the SSU ofmaize RuBisCO and the bacterial EPSPS gene.

Transit peptide of the SSU of sunflower RuBisCO:

The fragment is derived from the cDNA isolated by Waksman and FreyssinetNucl. Acids Res. 15:1328 (1987). An SphI site was created at thecleavage site of the transit peptide according to the method of Zollerand Smith Methods in Enzymology 154:329 (1984). The transit peptide ofthe SSU of sunflower RuBisCO thus obtained is a 171-bp EcorI-SphIfragment.

Transit peptide of the SSU of sunflower RuBisCO/sequence of 22 aminoacids of the mature part of the SSU of maize RuBisCO/AroA gene fusion:

The construct containing the transit peptide of the SSU of maizeRuBisCO/sequence of 22 amino acids of the SSU of maize RuBisCO was cutwith EcoRI-SphI and then ligated with the 171-bp EcoRI-SphI fragmentcorresponding to the transit peptide of the SSU of sunflower RuBisCO. Aresulting construct exhibits a substitution of the EcoRI-SphI fragmentsand is a translational fusion “transit peptide of the SSU of sunflowerRuBisCO/sequence of 22 amino acids of the SSU of maize RuBisCO/AroAgene.

The EcoRI-SalI fragment was ligated with the SalI-SstI fragmentcontaining the 3′ nos sequence and the right end of the T-DNA. Theresulting EcoRI-SstI fragment, comprising “transit peptide of the SSU ofsunflower RuBisCO/sequence of 22 amino acids of the SSU of maizeRuBisCO/AroA gene/3′ nos/T-DNA right end”, is substituted for theEcoRI-SstI fragment containing the right end of the T-DNA of the plasmid150 A alpha 2 containing the double CaMV promoter. The transcriptionalfusion “double CaMV/transit peptide of the SSU of sunflowerRuBisCO/sequence of 22 amino acids of the SSU of maize RuBisCO/AroAgene/3′ nosy” in the vector 150 A alpha 2 was called pRPA-BL 294.

“Transit peptide of the SSU of sunflower RuBisCO/sequence of 22 aminoacids of the SSU of maize RuBisCO/transit peptide of the SSU of maizeRuBisCO/AroA gene” fusion:

The above construct is cut with NcoI-HindIII, releasing the AroA gene.Next it is ligated with a 1.5 kbp NcoI-HindIII fragment containing the“transit peptide of the SSU of maize RuBisCO/AroA gene” fusion. Aresulting construct exhibits a substitution of the NcoI-HindIIIfragments and is a translational fusion “transit peptide of the SSU ofsunflower RuBisCO/sequence of 22 amino acids of SSU of maizeRuBisCO/transit peptide of the SSU of maize RuBisCO/AroA gene”.

The EcoRI-SalI fragment was ligated with the SalI- SstI fragmentcontaining the 3′ nos sequence and the right end of the T-DNA. Theresulting EcoRI-SstI fragment comprising “transit peptide of the SSU ofsunflower RuBisCO/sequence of 22 amino acids of the SSU of maizeRuBisCO/transit peptide of the SSU of maize RuBisCO/AroA gene/3′nos/T-DNA right end” is substituted for the EcoRI-SstI fragmentcontaining the right end of the T-DNA of the plasmid 150 A alpha 2containing the double CaMV promoter. The transcriptional fusion “doubleCaMV/transit peptide of the SSU of sunflower RuBisCO/sequence of 22amino acids of the SSU of maize RuBisCO/transit peptide of the SSU ofmaize RuBisCO/AroA gene/3′ nos” in the vector 150 A alpha 2 was calledpRPA-BL 410.

All the constructs were obtained by substituting the double CaMVpromoter region of the pRPA-BL 410 construct obtained above, which iscut in EcoRI-HindIII, with a HindIII-EcoRI fragment containing thevarious promoter elements which follow:

A) Single H4 A748 histone promoter:

An XhoI-XhoI fragment of about 1 kbp containing the Arabidopsis thalianaH4 748 histone promoter (Plant Mol.Biol. Vol 8, 1987 p 179-191) Bwasligated with SalI-cut pUC 19. This clone was then cut with XbaI treatedwith Klenow polymerase and EcoRI treated with Klenow polymerase and thenligated under conditions which promote recircularisation. TheHindIII-EcoRI fragment containing the histone promoter was substitutedfor the HindIII-EcoRI fragment containing the pRPA-BL 410 double CaMVpromoter.

The resulting clone contains the “H4 histone promoter/transit peptide ofthe SSU of sunflower RuBisCO/sequence of 22 amino acids of the SSU ofmaize RuBisCO/transit peptide of the SSU of maize RuBisCO/AroA gene/nos”and was called pRPA-BL 498.

B) Single H4 777 histone promoter

An XhoI fragment of about 1 kbp containing all the proximal 5′ regionsof the Arabidolpsis thaliana H4 777 histone gene cf Plant Mol. Biol. Vol8, 1987 p 179-191 was cloned at the SalI site of the plasmid pUC 19 withan orientation such that the sites of the pUC 19 polylinker from XbaI toEcoRI were situated downstream of the site of initiation oftranscription.

Transit region of the SSU of sunflower RuBisCO/AroA fusion AnEcoRI-HindII fragment of the cDNA of the SSU of sunflower RuBisCOisolated by Waksman and Freyssinet (1987) encoding the transit peptideand 30 amino acids of the mature protein was cloned at the EcoRI andSmaI sites of pUC 18.

An EcoRI-XbaI fragment was isolated and inserted upstream of the AroAgene of pMG 34-2 (provided by Calgene) at the EcoRI and XbaI sites. Inorder to put the 2 coding regions in frame, the EcoR-HindIII insert ofthis clone was inserted in M13 mp19 at the EcoRI and HindIII sites.

The replicative form of the DNA of this clone was linearised with XbaIand then treated with Vigna radiata nuclease. After ligation underconditions promoting recircularisation and transformation, the DNA ofsome clones was analysed by sequencing. One of the clones obtainedproduces a translational fusion between the SSU element of RuBisCO andthe AroA fragment, the initiation codon ATG of the latter being lost.The sequence of the junction between these two elements is as follows:

GAC GGGGATCCGGAA

SSU linker AroA.

This fusion therefore comprises in the direction of translation: transitpeptide of the SSU of sunflower RuBisCO/30 amino acids of the maturepart of the SSU of sunflower RuBisCO/3 amino acids corresponding to theintermediate linker/AroA.

Construction of pRPA - BL - 240

The plasmid comprising the promoter of the H4 777 histone gene describedabove was cut with XbaI, treated with Klenow polymerase and the fragmentresulting from the HindIII recut isolated. The plasmid containing thesunflower transit peptide/AroA fusion described above was cut withEcoRI, treated with Klenow polymerase and the fragment resulting fromthe HindIII recut isolated. These two fragments were ligated in thepresence of HindIII-cut pUC 19. One of the resulting clones has thefollowing structure: histone promoter/region encoding the transitpeptide of the SSU of sunflower RuBisCO/zone encoding 30 amino acids ofthe mature part of the SSU of sunflower RuBisCO/zone encoding 3 aminoacids corresponding to the intermediate linker/zone encoding AroA.

This clone was cut with SalI, treated with Klenow polymerase and then,after recutting with HindIII, the isolated fragment was ligated with theplasmid containing the polyadenylation region, cut with BamHI, treatedwith Klenow polymerase and then recut with HindIII.

One of the resulting clones has the following structure: histonepromoter/zone encoding the transit peptide of the SSU of sunflowerRuBisCO/zone encoding 30 amino acids of the mature part of sunflowerRuBisCO/zone encoding 3 amino acids corresponding to the intermediatelinker/zone encoding AroA/3′ nos.

The above plasmid was linearized with HindIII and inserted at the uniqueHindIII site of the vector pRPA-BL 127 constructed in the followingmanner: the vector pCGN 1152 possessing a hygromycin-resistant geneunder the control of the mannopine synthase gene and the terminator ofthe Tm1 gene (provided by Calgene) was digested with HindIII andreligated to itself, leading to the creation of a unique HindIII cloningsite.

One of the resulting clones comprises the following transcriptionalfusion, from the left end of the T-DNA of the plasmid pRPA-BL 127:histone promoter/transit zone of the SSU of sunflower RuBisCO/AroA/3′nos, and was called pRPA-BL-240.

C) Duplication of the histone promoter (=double histone promoter).

The H4 histone promoter in pUC 19 described above was digested withNdeI, treated with Klenow polymerase and then recut with HindIII. Thefragment removed was substituted by a HindIII—EcoRV fragment purified bydigesting the plasmid carrying the H4 histone promoter. One of theclones obtained has the following structure: 5′ region of the H4promoter of 530 bp from HindIII to NdeI, duplicated region correspondingto the two directly repeated fragments of 330 bp from NdeI to EcoRV, 3′region of the H4 promoter of 140 bp from EcoRV to EcoRI.

The HindIII-EcoRI fragment containing the double histone promoter wassubstituted for the HindIII-EcoRI fragment containing the double CaMV ofpRPA-BL 410. The resulting clone contains a “double histonepromoter/transit peptide of the SSU of sunflower RuBisCO/sequence of 22amino acids of the SSU of maize RuBisCO/maize transit peptide/AroAgene/nos” and was called pRPA-BL 488.

D) Hybrid double CaMV/histone promoter:

The H4 A748 histone promoter, in the HindIII-EcoRI cassette describedabove (single H4 A748 histone promoter), is cut with AccI, treated withKlenow polymerase and then cut with EcoRI. The resulting fragment of 580bp is ligated with the double CaMV promoter which is cut withEcoRV-EcoRI. The resulting clone comprising the double CaMV 3′ part ofthe histone promoter fusion is cut with HindIII-EcoRI and substitutedfor the HindIII-EcoRI fragment of pRPA-BL 410 comprising the doubleCaMV. The resulting construct comprises a “double CaMV promoter/3′ parthistone promoter/transit peptide of the SSU of sunflowerRuBisCO/sequence of 22 amino acids of the SSU of maize RuBisCO/transitpeptide of the SSU of maize RuBisCO/AroA gene/nos” and was calledpRPA-BL 502.

EXAMPLE 2

RESISTANCE OF THE TRANSFORMED PLANTS

1. Transformation:

The vector is introduced into the nononcogenic agrobacterium strain EHA101 (Hood et al., 1987) carrying the cosmid pTVK 291 (Komari et al.,1986). The transformation method is based on the procedure of Horsh etal. (1985).

2. Regeneration:

The regeneration of the tobacco PBD6 (source SEITA France) using foliarexplants is carried out on a Murashige and Skoog (MS) basic mediumcontaining 30 g/l of sucrose and 200 g/ml of kanamycin. The foliarexplants are removed from greenhouse- or in vitro-grown plants andtransformed according to the foliar disc method (Science 1985, Vol. 227,p. 1229-1231) in three successive stages: the first comprises theinduction of shoots on an MS medium supplemented with 30 g of sucrosecontaining 0.05 mg of naphthylacetic acid (ANA) and 2 mg/l ofbenzylaminopurine (BAP), for 15 days. The shoots formed during thisstage are then developed by culturing on an MS medium supplemented with30 g/l of sucrose, but not containing hormone, for 10 days. Thedeveloped shoots are then removed and they are cultured on ahalf-diluted MS planting medium containing half the content of salts,vitamins and sugars and not containing hormone. After about 15 days, thedeeply-rooted shoots are placed in soil. Untransformed tobacco PBD 6plants are grown up to the same stage as controls.

3. Measurement of the glyphosate tolerance:

The best plants obtained from the regeneration of transformed tobaccosare selected under a greenhouse after treating the plants, at the 5-leafstage, by spraying with an aqueous suspension of glyphosate at a dosecorresponding to 0.6 kg/ha of active substance. The plants selected areself-fertilised and the seeds are sown on an MS medium supplemented with30 g/l of sucrose and 200 micrograms/ml of kanamycin. The seeds obtainedfrom plants having a segregation ratio of ¾ of resistant plants-¼ ofsensitive plants were kept. Molecular hybridization analysis enabled theplants having integrated only one copy of the T-DNA to be determined.These plants were taken to the heterozygote or homozygote state bycrossbreeding and selection. These plants were sown under naturalconditions, in the open, and sprayed at the 5-leaf stage with a dose of0.8 kg/ha of glyphosate (Round Up). The results correspond to theobservation of phytotoxicity indices taken at the end of 30 days afterthe treatment. Under these conditions, it is observed that the plantstransformed with the constructs exhibit an acceptable tolerance (pRPA-BL240 and 410) or even a good tolerance (pRPA-BL 488 and 502) whereas thecontrol plants are completely destroyed.

These results clearly show the improvement brought by the use of achimeric gene according to the invention for the same gene encoding theglyphosate tolerance.

The transformed plants according to the invention may be used as parentsfor producing lines and hybrids having an increased tolerance toglyphosate.

EXAMPLE 3

Spring colzas, Westar cultivar, resistant to glyphosate, were obtainedusing the method of BOULTER et al., 1990 (Plant Science, 70: 91-99),with the constructs pRPA-BL 488 (dH-At-PTO-aroA-nos), pRPA-BL 502(CaMV/H-At-PTO- aroA-nos). These plants were resistant to a greenhousetreatment with glyphosate at 400 g a.s/ha (grams of active substance perhectare, a treatment which destroys nontransgenic plants.

What is claimed is:
 1. A plant cell containing a chimeric gene, thechimeric gene encoding a transit peptide region comprising, in thedirection of translation, at least one transit peptide of aplastid-localized enzyme, a partial sequence of an N-terminal maturepart of a plastid-localized enzyme, and a second transit peptide of aplastid-localized enzyme.
 2. The plant cell of claim 1, wherein thetransit peptides and partial mature sequence are from sunflower ormaize.
 3. The plant cell of claim 2, wherein the chimeric gene furthercomprises a promoter region comprising at least one fragment of anArabidopsis H3 or H4 histone gene promoter.
 4. A plant which contains inits genome a chimeric gene encoding a transit peptide region comprising,in the direction of translation, at least one transit peptide of a plantgene which encodes a plastid-localized enzyme, a partial sequence of anN-terminal mature part of the plastid-localized enzyme, and a secondtransit peptide of a plastid-localized enzyme.
 5. The plant of claim 4,wherein the chimeric gene further comprises a promoter region comprisingat least one fragment of an Arabidopsis H3 or H4 histone gene promoter.